We describe here the application of a simple and efficient method to control an endogenous bacterial contamination of in vitro shoot cultures of apple through successive subcultures of shoot apices on media with cefotaxime. Endogenous bacteria are frequent in cultures derived from explants taken from adult trees grown in the field and affect both shoot growth and organogenesis as in the adventitious regeneration of shoots and roots. We used apple shoots cv. “Douce de Djerba” cultured in vitro that showed bacterial growth at the base of the shoots. While 1 h treatments with 0.1% or 1.0% sodium hypochlorite were harmful and ineffective, the addition of sterile cefotaxime to autoclaved culture medium (a modified MS medium) at 150 mg l-1 improved both shoot length and leaf expansion inhibiting bacterial growth. After the third subculture on medium with cefotaxime no visible bacterial growth was detected and shoots grown on MS without cefotaxime did not show any bacterial growth both in the culture medium and in PYGA plates. Length of cefotaxime-treated shoots was twofold that of untreated shoots (32.7 ± 2.71 mm vs. 15.95 ± 1.48 mm respectively). The method described here would allow the control of endogenous bacteria that are sensitive to cefotaxime in in vitro cultures, removing a source of uncontrolled variability that could affect different studies.